waters Co-occurrence of Dinophysis tripos and pectenotoxins in Argentineanshelf Harmful Algae

Co-occurrence of Dinophysis tripos and pectenotoxins in Argentinean shelf waters

Elena Fabro

^a,b

, Gasto´n O. Almandoz

^a,b

, Martha E. Ferrario

^a,b

, Mo´nica S. Hoffmeyer

^b,c,d

, Rosa E. Pettigrosso

^e

, Roma´n Uibrig

^d

, Bernd Krock

^f,

*

aDivisio´nFicologı´a,FacultaddeCienciasNaturalesyMuseo,UniversidadNacionaldeLaPlata,PaseodelBosques/n,B1900FWALaPlata,Argentina

bConsejoNacionaldeInvestigacionesCientı´ficasyTe´cnicas(CONICET),Av.Rivadavia1917,1033BuenosAires,Argentina

cFacultadRegionaldeBahı´aBlanca,UniversidadTecnolo´gicaNacional,11deAbril461,B8000LMIBahı´aBlanca,Argentina

dInstitutoArgentinodeOceanografı´a(CCTBBCONICET),CaminoLaCarrindangakm7.5,B8000FWBBahı´aBlanca,Argentina

eDepartamentodeBiologı´a,Bioquı´micayFarmacia,UniversidadNacionaldelSur,SanJuan670,8000Bahı´aBlanca,Argentina

fAlfredWegenerInstitut-HelmholtzZentrumfu¨rPolar-undMeeresforschung,ChemischeO¨kologie,AmHandelshafen12,27570Bremerhaven,Germany

1. Introduction

ThegenusDinophysisEhrenbergisrepresentedbymorethan 120speciesofmarinedinoflagellates,includingphototrophicand mixotrophicspecies(Hastrup-JensenandDaugbjerg,2009;Go´mez etal., 2011). Ofthese,10 toxic speciesare known asthemain source of okadaic acid and its analogs, causative of diarrheic shellfishpoisoning(DSP)inhumans(Regueraetal.,2014).Afew hundredcells perliter areenough togenerate intoxications in humansby consumingcontaminatedshellfish (Yasumoto etal., 1980).Uptodate,dinoflagellatespeciesofthegenusDinophysis have been recently recognized as the only known source of pectenotoxins(PTX),alargefamilyoflipophilictoxinsoriginally associated with the DSP toxin complex (Reguera et al., 2014).

ToxicologicalstudiesindicatethatPTXarenotdiarrheagenicafter oral administration to laboratory rodents, but PTX-1 is highly

hepatotoxicand theotheranalogs areassumed tohavesimilar effects (Terao et al., 1986). Reported lethal doses (LD50) by intraperitoneal injection (i.p.) in mice for PTXs are detailed in Domı´nguezetal.(2010).Cytotoxicityindifferenthumancancer linesandinductionofapoptosisinratandsalmonhepatocyteshas beenalsoattributedtosomePTX (Domı´nguezetal.,2010). The existence of different published results reported by different groupshasraisedacontroversyaboutthediarrheicactivityand oraltoxicityofPTX.

Unlike DSP toxins as okadaic acid, which are known to accumulate through the food web and concentrate in somatic tissuesofzooplanktonicorganisms(Teegarden&Cembella,1996;

Suzukietal.,1998;Maneiroetal.,2000;Testeretal.,2000),asfar as we know there is no evidence of PTX bioaccumulation in zooplankton, although copepod grazing on Dinophysis spp.

(Maneiro et al., 2002; Kozlowsky-Suzuki et al., 2006) is well known.Pectenotoxin-2wasdetectedincopepodsincubatedwith Dinophysisspp.(Seta¨la¨ etal.,2009),buttheabsenceofthistoxinin individualcopepodsisolatedfromfieldzooplanktonsamplesled theauthorstoconcludethatatoxintransferthroughthefoodweb ARTICLE INFO

Articlehistory:

Received21August2014

Receivedinrevisedform18December2014 Accepted18December2014

Availableonline

Keywords:

Lipophilictoxins LC–MS/MS Dinophysistripos Morphologicalvariability Zooplanktonbioaccumulation SouthAtlantic

ABSTRACT

ThespeciesDinophysistriposisawidelydistributedmarinedinoflagellateassociatedwithdiarrheic shellfishpoisoning(DSP)events,whichhasbeenrecentlyidentifiedasapectenotoxin(PTX)producer.In twosamplingexpeditionscarriedoutduringaustralautumns2012and2013alongtheArgentineSea (38–568S),lipophilicphycotoxinsweremeasuredbytandemmassspectrometrycoupledtoliquid chromatography(LC–MS/MS)insize-fractionatedplanktonsamplestogetherwithmicroscopicanalyses ofpotentiallytoxicphytoplankton.PTX-2,PTX-11andPTX-2sawererecurrentlydetectedinthe50–

200mmfractions,inassociationtoD.tripos.PTX-2wasalsowidelydistributedamongthe20–50mm fractions,mostlyrelatedtoDinophysisacuminata.Okadaicacidoritsanalogswerenotdetectedinany sample.ThisisthefirstreportofD.triposrelatedtoPTXintheArgentineSeaandthefirstrecordofPTX- 11and PTX-2saforthisarea.The morphologicalvariabilityofD. tripos,includingthepresenceof intermediate,smallanddimorphiccells,isdescribed.Also,themicro-andmesoplanktonicpotential grazersofDinophysisspp.wereexplored.

ß2015ElsevierB.V.Allrightsreserved.

* Correspondingauthor.Tel.:+4947148312055;fax:+4947148311425.

E-mailaddress:bernd.krock@awi.de(B.Krock).

ContentslistsavailableatScienceDirect

Harmful Algae

j our na l ho me p a ge : w ww . e l se v i e r . com / l oc a te / h a l

http://dx.doi.org/10.1016/j.hal.2014.12.005 1568-9883/ß2015ElsevierB.V.Allrightsreserved.

could not be proven (Seta¨la¨ et al., 2011). However, PTX metabolizedbyenzymatichydrolysishavebeenfoundinmussels (Milesetal.,2004b;Wilkinsetal.,2006).

Pectenotoxinsarecyclicpolyetherlactonesthatdifferstructur- allyfromeach otherbydifferentdegreesofoxidation, different arrangementsofthespiroketalring system,and openingof the large lactone ring (Quilliam, 2003). These toxins are globally distributedandrecurrentlyfoundatthesoutheastPacificcoastof Chile(Blancoetal.,2007;Krocketal.,2009;Trefaultetal.,2011).In Argentineanwaters,thepresenceofPTX-2wasrecentlydetected intheBuenosAiresprovincecoast(Montoyaetal.,2013),andin theSanJorgeGulf(B.Krock,unpubl.data).

IntheIOC-UNESCOreferencelistoftoxicmicroalgae(Zingone&

Larsen,2014)DinophysistriposGourretisoneofthe10Dinophysis species included. Detection of DTX-1 in picked cells from Kesennuma Bay (Japan) using liquid chromatography with fluorescence detection (LC-FLD) was the first record of toxin productionbythisspecies(Leeetal.,1989).However,thesetoxins werenotdetectedlaterinD.triposisolatesfromJapananalyzedby liquidchromatography–tandemmass spectrometry(LC–MS/MS) (Suzukietal.,2009).Morerecently,thepresenceofPTX-2inD.

triposwasdetectedforthefirsttimeinfieldsamplesandcultures fromGalicianRı´as alsousing LC–MSanalyses(Rodrı´guezetal., 2012).Inaccordance,theproductionofPTX-2andtoamuchlesser extentof DTX-1 wasrecentlyproven inJapanese culturesof D.

triposusingLC–MS/MS(Nagaietal.,2013).

ThespeciesDinophysistriposiswidelydistributedinsubtropical andtropicalwaters,butcanalsobeobservedincolderregionssuch asthe northernNorwegian Sea and subantarctic waters of the SouthAtlantic(Johnsen&Lømsland,2010).IntheArgentineSea,it isknowntooccurbetween36and558S(Balech,2002),andithas beenfrequentlyobservedalongnortherncoastalwatersofBuenos AiresProvince(36–378S),reachingdensitiesupto310²cellL¹ (Saretal.,2010).Thedistinctivecellshapeandsize,includingthe presence of two posterior hypothecal projections, allow the differentiation of D. tripos from other related species (Balech, 1988;Larsen&Moestrup,1992).However,thepresenceofatypical intermediateandsmallcellformsofD.tripos,originallydescribed asDinophysisdiegensisvar. curvataKofoid,aswellasdimorphic formsresultof‘‘depauperating’’celldivisioncanalsobeobserved (Reguera&Gonza´lez-Gil,2001;Rodrı´guezetal.,2012).

Withthepurposeofstudyingpotentiallytoxicmicroalgaeand their toxins in Argentinean shelf waters,two expeditions took place covering an extended area (38–568S). In addition, the occurrence of possible predators of Dinophysis tripos was also analyzedinordertostudypossibletoxintransferthroughthefood web. In this work, we describe the recurrent finding of pectenotoxinsassociatedwiththedinoflagellateD.tripos,provid- ingthefirstreportlinkingthisspecieswithtoxinsintheArgentine Sea. Adescription ofdifferent morphologicalforms of D.tripos observedintheareaisalsopresented.

2. Materialsandmethods

2.1. Sampling

ThecontinentalshelfwatersoftheArgentineSeaweresampled duringtwoexpeditions.Thefirstonewascarriedoutonboardthe R/V‘‘PuertoDeseado’’fromMarch30thtoApril14th,2012.Atotal of46stationsweresampledbetween388and558S(Fig.1).The secondwasperformedontheR/V‘‘BernardoHoussay’’fromMarch 11th to March 22nd, 2013 with 24 sampling points located between398and438S.Itconsistedoftwolegs,K1(eightstations) andK2(16stations).

Planktonsampleswerecollectedbyverticalnettowsthrough theupper20mofthewatercolumnwitha20

m

m-meshNitexnet

forbothplanktonandphycotoxins analysis.Each nethaulwas takenupto1Lwith0.2

m

mfilteredseawater,ofwhich20mLand 100mLwerefixedwithLugol’ssolutionduringexpedition1and 2respectively,forspeciesdeterminationandenumeration.The restwassequentiallyfilteredthroughNitexmeshof200,50and 20

m

mbygravityfiltrationandsplitintoaliquotsforextractionof toxins.

2.2. Phytoplanktonanalysis

CellabundanceofDinophysisspp.innettowconcentrateswas determined by counting 1mL of Lugol fixed samples using Sedgewick-Rafterchambers(LeGresley&McDermott,2010)with aninvertedmicroscope(LeicaDMILLED).Furthermorphological examinationwasmadewitha phasecontrast/differentialinter- ference contrast Leica DM2500 microscope equipped with a DFC420Ccamera,andascanningelectronmicroscope(FEIQuanta FEG200,Eindhoven,Netherlands).

2.3. Zooplanktonanalysis

Samples(20

m

m-meshnet) fromthe legK2 of expedition2 (n=16)werealsoanalyzedformicrozooplankton(20–200

m

mand

>200

m

minsize)analysis.Thelatterwereidentifiedtothelow

taxonomicpossiblelevelandenumeratedfollowingtheUtermo¨hl method(Utermo¨hl,1931)usingappropriateliterature(Boltovskoy, 1981;Balech,1988;Montagnesetal.,1988;Montagnes&Lynn, 1991; Montagnes&Taylor, 1994; Agatha &Riedel-Lorje,1997;

Alder, 1999; Petz, 1999; Kogan, 2005). Each sample was well homogenizedand10mLallowedsettlingfor24h.Afterthat,all organismsinthebasechamberwereidentifiedandcountedunder a Nikon Eclipse TS100 inverted microscope. Some possible predatorsofDinophysisspp.presentinsamples(includingthose

of >200

m

m of size) wereexamined in more detail under the

Fig.1.Mapofthestudyareashowingthelocationofsamplingpoints.

mentionedinvertedmicroscope,aNikonSMZ1500stereomicro- scope and a Nikon Eclipse 80i microscope sometimes using methylene bluestain togetbettercontrast. Imagesfromthem wereobtainedusing a NikonDXM 1200Fdigital camerawhich were later processed with Image Pro plus software (Media Cybernetics).

2.4. Toxinanalysis

Cell pellets from the plankton net tow size fractions were collectedbycentrifugation(3220g,15minat48C),suspended in500

m

Lmethanol,andsubsequentlyhomogenizedwith0.9gof lysing matrix D by reciprocal shaking at maximum speed (6.5ms¹) for 45s in a Bio101 FastPrep instrument (Thermo Savant, Illkirch, France). After homogenization, samples were centrifugedat16,100gat48Cfor15min.Thesupernatantwas transferredtoaspin-filter(0.45

m

mpore-size,MilliporeUltrafree, Eschborn,Germany)andcentrifugedfor30sat800g,followed bytransfer toautosamplervials. Analysisofmultiplelipophilic toxins was performed by liquid chromatography coupled to tandem mass spectrometry(LC–MS/MS), as described in Krock etal.(2008).

3. Results

3.1. Planktonandphycotoxinanalysis 3.1.1. Expedition1

ThemostfrequentDinophysisspp.observedalongexpedition1 wasDinophysisacuminata(37–45

m

m),occurringin82%ofthe stations in abundances from 110³ to 16810³cellsNT¹. However, the maximum Dinophysis abundance (20.710⁴cellsNT¹) was reached by Dinophysis tripos (70–

90

m

m)atstationI50(408S),whilenootherDinophysisspecies wasobservedat thissamplingpoint.In fact,thepresenceofD.

triposduringtheexpedition1wasalmostconfinedtostationI50, sinceitappearedinonlytwomorestationsinverylowabundances (1–210³cellsNT¹)(Fig.2A).

AnalysisofphycotoxinsrevealedaveryhighabundanceofPTX- 2sa(2396ngNT¹)inthe50–200

m

mfractionsizeatstationI50

(Fig.2B).Pectenotoxin-2secoacidappearedinthissizefractionin onlyfivemoresamplesbutinlowerconcentrations(from104to 266ngNT¹). Also low concentrations of PTX-2 (from 106 to 578ngNT¹)weredetectedinfoursamplesinthis fractionsize (Fig.2B).In contrast, inthe 20–50

m

msizefractions, themost distributedtoxinwasPTX-2,occurringin61%ofthestations(data not shown). In this fraction, also PTX-2sa was detected in 15 samplesintraceconcentrations(<35ngNT¹)andintwosamples inlowvalues(56and78ngNT¹).Pectenotoxin-2secoacidwas the only toxin present in the >200

m

m size fraction, with a maximumvalueatstationI50(107ngNT¹).Nookadaicacidorits analogsweredetectedinanysizefraction.

3.1.2. Expedition2

ThemostcommonDinophysisspeciesobservedalongexpedi- tion2wasDinophysistripos(79–100

m

m),beingpresentin12 stationslocatedbetween418and438S(legK2).Itrepresentedan averageof83%ofthetotalabundanceofthegenus,withdensities ranging between 1110³cellsNT¹ and 380010³cellsNT¹ (Fig.3A).ThespeciesDinophysisacuminata(25–45

m

m)wasalso presentinnineoftotalsamples,butinlowerabundances,from1to 2210³cellsNT¹.

ToxinanalysesshowedthepresenceofthreePTX inthe50–

200

m

msizefractions:PTX-2,PTX-11andPTX-2sa.Thelatterwas themostabundanttoxin,beingpresentin50%ofthesamplesand confined to the same 12 stations where Dinophysis tripos was found,whereasPTX-2andPTX-11weredetectedin46%and21%of stations,respectively(Fig.3B).ThemaximumlevelsofPTX-2sa, PTX-2andPTX-11were3002,1317and512.5ngNT¹respectively (Table1).Inthe20–50

m

msizefractionsonlytraceconcentrations ofthethreetoxinswerefound;PTX-2appearedat12stations,PTX- 2saat10stations,andPTX-11atonly1station(datanotshown).In

the>200

m

msizefractions,PTX-2andPTX-2saweredetectedalso

intraceconcentrationsintwoandninesamplesrespectively,but PTX-2sa also appeared in one sample showing a higher value (630ngNT¹).

High and significant(p<0.05)Spearman correlations coeffi- cientswerefoundbetweenDinophysistriposabundanceandPTX-2 (r=0.91),andPTX-2sa(r=0.96)concentrations.Nookadaicacidor itsanalogsweredetectedinanysizefraction.

Fig.2.DistributionofPTXsconcentrationinthe50–200mmfractionsizeandD.triposabundances,duringthefirstexpeditionintheArgentineSea.

Zooplankton analysesrevealedthepresenceofheterotrophic dinoflagellates, tintinnids, aloricate ciliates, different marine invertebratelarvae,cladocerans as wellas nauplius larvae and copepodites of cyclopoid, calanoid and harpacticoid copepods.

Takingintoaccounttheirsize,smallandmediumdinoflagellates, ciliates, some eggs and first nauplii of copepods belonged to microzooplankton (<200

m

m size fraction), while the large heterotrophicdinoflagellateNoctilucascintillans,copepodnauplii,

copepoditesandsomeadults,cladoceransandinvertebratelarvae belongedtomesozooplankton(>200fraction).Accordingtobody measures,thepotentialpredatorsofDinophysisspp.(e.g.,mainly Dinophysistriposwhichwasthedominantspeciesofthisgenusin samples)inthe20–200

m

msizefractionandtheirabundances,are presented in Table 2. They belong to dinoflagellates, aloricate ciliates, and tintinnids, all microzooplanktonic groups. None of thesetaxashowedadistributionpatternrelatedtothatofPTXin Fig.3.DistributionofPTXsconcentrationinthe50–200mmfractionsizeandD.triposabundances,duringthesecondexpeditionintheArgentineSea.

Table1

Co-occurrenceofpectenotoxins(PTXs)andD.tripos,andestimatedtoxincellquotasinthe50–200mmsizefractionsofnetsamplescollectedduringthesecondexpeditionin theArgentineSea.

Sample PTX2sa (ngNT¹)

PTX2 (ngNT¹)

PTX11 (ngNT¹)

Total (ngNT¹)

Dinophysistripos (cells10³NT¹)

PTX2sa (pgcell¹)

PTX2 (pgcell¹)

PTX11 (pgcell¹)

Total (pgcell¹)

2K2 27.0 0.0 0.0 27.0 13 2.07 0.00 0.00 2.07

3K2 560.7 6.9 0.0 567.6 273 2.05 0.03 0.00 2.08

4K2 2057.4 55.9 0.0 2113.3 1180 1.74 0.05 0.00 1.79

5K2 1069.7 280.6 245.1 1595.4 1570 0.68 0.18 0.16 1.02

6K2 1282.4 639.3 459.2 2380.9 1970 0.65 0.32 0.23 1.21

8K2 3001.9 100.4 0.0 3102.3 3350 0.90 0.03 0.00 0.93

9K2 2441.2 1316.9 512.5 4270.7 3800 0.64 0.35 0.13 1.12

10K2 1505.3 229.5 90.5 1825.3 1133 1.33 0.19 0.08 1.61

13K2 414.5 238.8 48.1 701.3 405 1.02 0.06 0.12 1.73

13bK2 323.1 123.7 0.0 446.8 295 1.10 0.04 0.00 1.51

20K2 81.5 42.5 0.0 124.0 170 0.48 0.03 0.00 0.73

21K2 68.1 3.4 0.0 71.6 11 6.20 0.03 0.00 6.51

Table2

AbundancesofpotentialpredatorsofD.tripospresentinthe20–200mmsizefractionsduringthelegK2ofexpedition2intheArgentineSea.

Station/cellsNT¹ 1K2 2K2 3K2 4K2 5K2 6K2 8K2 9K2 10K2 13K2 13bK2 20K2 21K2 27K2 28K2 29K2

Gyrodiniumspp. 0 0 0 0 0 0 0 0 6195 0 3640 0 0 0 0 0

Tiarinafusus 0 0 7735 13,350 5005 30,030 3540 0 1035 27,880 28,220 620 0 0 670 1098

Strombidinopsisacuminata 0 0 0 1335 0 0 0 0 0 410 415 0 0 0 0 122

Strombidinopsiselongata 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 61

Cyclotrichiumgigas 0 276 910 1335 0 1365 0 0 0 1230 1245 0 940 0 0 61

Codonellopsisobesa 0 4920 840 0 0 0 0 0 0 0 0 0 0 0 1530 20,235

Favellataraikaensis 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 570

Favellaehrenbergi 0 168,100 8400 2765 21,060 810 0 0 9292 5040 12,775 4095 840 1890 0 11,970

the 50–200

m

m size fraction. Copepod fecal pellets were also frequentlyobservedinthissizefractionofsamplesbutwerenot quantified.Inthe>200sizefractionDinophysisspp.grazersasN.

scintillansandotherpotentialonesasdifferentdevelopmentstages ofcopepodsappearedatallstations(Table3).

3.2. Dinophysistriposmorphology

Dinophysis tripos normal cells are easily distinguished from otherspeciesbytheirtypicalcellshape,whichincludestwobig posteriorprolongations,onedorsalandtheotherventral.Alsothe cell size is characteristic, ranging between 94 and 125

m

m.

Cingularlistsarewelldevelopedandtheleftsulcallistisbigger thantherightone(Balech,1988).

Besides the normal shape of Dinophysis tripos another two different forms, intermediate and small cells according to Rodrı´guez et al. (2012), were commonly observed along the secondexpedition.Thesetwomorphotypesdifferfromthetypical form in terms of lengthand dorso-ventraldepth of the larger hypothecalplates(Table4);developmentofdorsalhorn,absentin small cells and reduced in intermediate cells (Fig. 4); and areolation,markedinnormalcellsbutabsentinsmallcellforms (Fig.5).

Intermediateandsmallformswerepresentin9of12samplesin whichDinophysistriposwasrecorded.However,theyrepresented only 0.18–2.54% (average of 0.86%) of the total abundances of D.tripos.Ontheotherhand,inthethreesamplesinwhichonly normal cells were found, the abundance of D. tripos was considerablylow(maximumof17010³cellsNT¹).

Recently dividedvegetative cells,lacking theanteriororthe posteriorleftsulcallist,wereobservedinallsamplesthatcontain Dinophysistriposnormalcells(Fig.6A).Inaddition,dimorphiccells, result of depaupering division (Reguera & Gonza´lez-Gil, 2001), were found. They presented different hypothecalplates in the same individual,one ofthem withnormal appearanceand the otheronewithintermediateorsmallcellcharacteristics(Fig.6B andC).

4. Discussion

ThedinoflagellategenusDinophysisisconsideredastheonly sourceofPTXinmarinewaters(Regueraetal.,2014).SeveralPTX analogshavebeenfoundincultureconditions,fieldwatersamples andshellfish(Domı´nguezetal.,2010).ThespeciesDinophysisfortii wasthefirstoneidentifiedasa PTXproducer(Leeet al.,1989;

Draiscietal.,1996),butPTXhavebeenlaterfoundinDinophysis acuta(Suzukietal.,2003),Dinophysisnorvegica(Milesetal.,2004a;

Suzukietal.,2009),Dinophysisacuminata(MacKenzieetal.,2005), Dinophysiscaudata(Ferna´ndezetal.,2006)andDinophysistripos (Rodrı´guezetal.,2012;Nagaietal.,2013).

Inthepresentstudy,theoccurrenceofPTXinthe50–200

m

m size fractions wasobserved togetherwith thepresence of two potentially toxic Dinophysisspecies, Dinophysistripos and Dino- physisacuminata.However,cells ofD.acuminataranginginsize from39 to53

m

m(Balech,2002)andfrom25 to45

m

m(this Table3

Abundances(cellsNT¹)ofpotentialpredatorsofD.tripospresentinthe>200mmfractionsizeduringthelegK2ofexpedition2intheArgentineSea,andPTX-2sa concentration(ngNT¹)inthesamefraction.

Sample 1K2 2K2 3K2 4K2 5K2 6K2 8K2 9K2 10K2 13K2 13bK2 20K2 21K2 27K2 28K2 29K2

PTX2sa(ngNT¹) 0 0 18 7 4 8 630 50 17 23 4 47 0 0 0 0

Noctilucascintillans (cellsNT¹)

0 0 0 0 60,750 22,680 51450 2975 15,488 0 1820 1365 0 68,040 8670 0

CopepoditesCyclopoida (cellsNT¹)

5000 11,480 3360 3950 15,390 17,010 6370 5100 8850 0 2555 780 0 1800 2550 7980

CopepoditesCalanoida (cellsNT¹)

13,000 27,880 15,120 15,010 33,210 38,070 14,210 17,000 24,485 15,120 25,550 7540 2520 18,720 7650 19,380 CopepoditesHarpacticoida

(cellsNT¹)

0 4100 10,080 0 0 0 0 0 0 0 0 0 2520 0 510 0

Table4

Length (L) and dorso-ventral depth (E) of the hypothecal plates of normal, intermediateandsmallcellformsofD.triposobservedintheArgentineSea.

Normalcells Intermediatecells Smallcells

Lrange(mm) 79–100 72–87 59–70

Erange(mm) 42–50 28–40 25–32

Laverage(mm) 94 79 65

Eaverage(mm) 45 34 28

Fig.4.Lightmicrographs(phasecontrast)ofthedifferentmorphologicalformsofD.triposfoundinfieldsamples,normal(A),intermediate(B)andsmallsizeddells(C).

study)areexpectedtobecollectedinthe20–50

m

msizefraction, butmostPTX weredetected inthe50–200

m

mfraction, which arguesagainstasubstantialcontributionofD.acuminatatothePTX detected.Inaddition,insixsamplesfromthesecondexpeditionin whichPTX-2sawasdetected,D.triposwastheonlytoxicspecies foundinanyofthesesizefractions.InfivesamplescontainingPTX- 2andintwowithpresenceofPTX-11D.triposalsowastheonly toxicspeciesfound.Incontrast,D.acuminatawasdetectedalways co-occurring with D. tripos in the samples with PTX from expedition2.

Pectenotoxin-11hasbeenreportedfromNewZealand(Suzuki etal.,2003),theGalicianRı´as,Spain(Pizarroetal.,2008),NorthSea (Krocketal.,2008),alongtheChileancoast(Blancoetal.,2007;

Krocketal.,2009; Trefaultetal., 2011)and fromtheBenguela Current,SouthAfrica(Pitcheretal.,2011),butthisisthefirstreport fortheArgentineanSea.Althoughthistoxinwasalreadyassociated withDinophysisacuta(MacKenzieetal.,2005;Suzukietal.,2006) andDinophysisacuminata(MacKenzieetal.,2005),ourdatadonot strongly supportDinophysis tripos being a producerof PTX-11, becausethere waslittle correlationbetween thetoxin and the species(r=0.67).Apossibleexplanationforthislowcorrelationis thatD.triposmightfeedontheoriginalproducerofthetoxinwhich couldbeD.acuminata,asitwaspresentinsomestations.Although dinoflagellatesareknowntofeedondiversetaxaincludingother

dinoflagellates(Tillman,2004),thisfeedingbehaviorhasnotbeen reportedfor D.tripos,but itcannotbeentirelyexcluded,asthe genusDinophysisisknowntobemixothrophicandthatgrowthof Dinophysis spp. are based on prey–predator interactions (Park etal.,2006;Nishitanietal.,2008).However,asD.triposwasthe onlytoxicspeciesintwosamplesfromexpedition2thatcontend PTX-11,cellquotas inthose sampleswerecalculated (0.08and 0.12pgcell¹).

ThefindingofPTX-2productionassociatedtoDinophysistripos intheArgentineSeaisinaccordancewithrecentexperimentaland fieldobservationsinotherregions(Rodrı´guezetal.,2012;Nagai et al.,2013). ThePTX-2cell quotaestimatedin ourfield study (0.03–0.35pgcell¹)isconsiderablylowerthanthoseobtainedin Japanesecultures(0.829.1pgcell¹atthebeginningofincuba- tionand1235.696.1pgcell¹attheendofincubation)byNagai etal.(2013).However,thehighPTXcell quotafound inJapanese culturescouldbearesultofmeasurementofintraandextracellular toxin content. Pectenotoxin-2 cell quotas obtained in field (45–

90pgcell¹) and culture (179–232pgcell¹) samples from the GalicianRı´as(Rodrı´guezetal.,2012)arealsohigherthancellquotas inourstudy.

VariabilityincelltoxincontentandPTX compositioncanbe highamongdifferentDinophysisspp.populations(Leeetal.,1989), evenwithinthesamespeciesandgeographicalarea,aswasfound Fig.5.Electronicmicrographsofsmall(A)andnormal(B)cellsofDinophysistripos,showingthedifferenthypothecalareolation.

Fig.6.Lightmicrographs(phasecontrast)ofrecentlydividednormalcelllackingtheanteriorportionoftheleftsulcallist(A)andrecentlydivideddimorphiccells(BandC).

Arrowsshowthesmall(B)andintermediate(C)partofthethecae.

between the coasts of the western and the eastern North Sea (Krocket al.,2008),and differentialproduction oftoxinsin the same species can vary considerably even within specimens collectedinthesamelocality(Ferna´ndezetal.,2006). Although variationintoxinproductionisverycommon,thecellquotasof PTX-2foundinthisstudyarestrikinglylow.TheaveragePTX-2cell quotaofDinophysistriposfoundduringthesecondexpeditionwas calculatedas0.1pgcell¹.Thisisless than10%ofPTX-2sa,cell quota1.6pgcell¹,whichisdominatingtheprofile.Thisisquite unusual,becausetodatePTX-2alwayshasbeenfoundtobethe mostabundant variantin PTX profiles of thegenus Dinophysis (Regueraetal.,2014andcitationstherein).However,itseemsthat, otherthanin mostDinophysisspp., D.tripos eithermetabolizes PTX-2 further to PTX-2sa or the biosynthesis of PTX-2 in this species is only a minor biosynthetic pathway and PTX-2sa is biosynthesizedindependentlyasthemainmetabolicproduct.

Pectenotoxin-2saiscommonlyassociatedtoenzymatichydro- lysisofPTX-2inmussels(Milesetal.,2004b;Wilkinsetal.,2006) or to enzymatic conversion after phytoplankton cell rupture (Ferna´ndezetal.,2006).Inthissense,associationbetweenPTX2-sa andDinophysiscaudatahasbeenpreviouslymentioned,butitwas interpretedasaresultofcellruptureduetosamplingmethodology ortransportationofthesamples(Takahashietal.,2007).However, this can be excluded in our case, because at stations from expedition1withdominanceofDinophysisacuminataPTX-2was themostabundantPTXinthe20–50

m

mfractionsizeandonly tracesofPTX-2saweredetected.Given thatsamplesfromboth expeditions(anddifferentsizefractions)weretreatedthesame way,itisextremelyunlikelythathydrolysisofPTX-2hasoccurred onlyinthosesamplescontainingDinophysistripos,butnotinthose containingD.acuminata.Another possibilitytoexplain PTX-2sa content is that D. tripos has been feeding on other Dinophysis species (e.g. D. acuminata) that produce PTX-2 and it has hydrolyzed the toxin through enzymatic digestive reactions (MacKenzie et al., 2012), but this has not been proven at the present. A third possible explanation would be copepod fecal pellets (i.e., assuming copepod grazing on Dinophysis species) (Maneiroetal.,2002)thatwerefrequentlyobservedoccurringin the20–200

m

msizefractions.However,thelackofquantitative data prevent this can be a proof. Finally, a fourth explanation wouldbethemicrograzerpredationonD.tripos.

SeveralpotentialpredatorsofDinophysiswereobservedinthe 20–200

m

msizefractionsofsomesamples(Table2).Amongthe possiblepredators,wepostulatetheciliateTiarinafususwhichhas been mentioned as consuming autotrophic dinoflagellates by Hansen(1991).Also,FavellaehrenbergiiandFavellaserrata,within tintinnids, which have also been signaled as predators on the

nanoflagellateAzadiniumspp.(Krocketal.,2009)andDinophysis acuminata (Maneiro et al., 2000) respectively. However, the associationbetweenthesepotentialpredatorsofDinophysistripos andPTX-2sawasnotsignificant(rmax=0.45).Alsoinsamples8K2 and9K2,withthehighestvaluesofPTX-2sa,potentialpredators werenotdetectedorpresentatreducedabundances(Table2).

Insummary,consideringthatthefourprevioushypothesesare notconclusivetoexplainthehighamountofPTX-2sainthe50–

200

m

msizefraction,andthehighcorrelationobservedbetween PTX-2saconcentrationandDinophysistriposabundances,ourdata suggestthisspeciesasaproducerofPTX-2sa,showingcellquotas rangingbetween0.48and6.2pgcell¹inthesecondexpedition, andahighervalue(11.6pgcell¹)atstationI50fromexpedition1.

ThisrelationbetweenPTX-2saandDinophysisspp.(8.16pgcell¹) waspreviouslyreportedfornaturalplanktonfoodsuspensionsrich in Dinophysis norvegica cells (Kozlowsky-Suzuki et al., 2006).

However,additionalfieldstudiesaswellascultureestablishment arerequiredtounambiguouslyelucidatethetoxinprofileofthis speciesandthepossibleproductionofPTX-2sabythisgenus,as wellastotestitsbioaccumulationinthefoodweb.

WiththepurposetoexploreapossibletransferofPTXthrough

the food web, the >200

m

m size fractions were analyzed for

putativeDinophysistripospredators.Numerousindividualsofthe largeheterotrophicdinoflagellateNoctilucascintillans,withbody diameter rangeof 200–750

m

maccordingto Satoetal. (2004), wereobservedcontainingpartialor completelyemptyD.tripos thecae(Fig.7),sopredationofN.scintillansonthisdinoflagellate seemsclear.ThisfindingagreeswiththereportofSatoetal.(2004) whichmentionsD.triposbeingpartofthedietofN.scintillansata costalfixedstation(EPEA,38828⁰S,57841⁰W)fromtheArgentine Sea shelf. These authors reported that N. scintillans is an opportunisticpredatorgrazingonthemostabundantmicroplank- ton items present in the water column such as copepod eggs, diatoms,tintinnidsandD.tripos.AssuchN.scintillanshasalsobeen recorded to feed on Dinophysis acuta and Dinophysis caudata (Escaleraetal., 2007)intheGalician Rı´as. Inaddition,this isa cosmopolitanheterotrophicdinoflagellateproducerofredtidesin numerous coastal areas in the world during spring-summer (Montani et al., 1998; Fonda-Umani et al., 2004). It has been classified as phagotrophic and its feeding mechanism is by engulfing preys (Hansen &Calado, 1999). The occurrence ofN.

scintillans as well as different copepodites would explain the observed levelsof PTX-2satoxinin the>200

m

mfractionsize.

However,itisnotpossibletoexcludethepossibilitythatsomeD.

tripos cells may have been retained on the200

m

mmesh and contributedtothePTX-2sapresenceinthisfraction.

MorphologicalanalysesofDinophysistriposrevealeddifferent morphotypesinalmostallpopulationsoftheArgentineanSea.This was already pointed for other populations, as formation of intermediateandsmallcellformsanddimorphiccellshavebeen recordedforanumberofspeciesofthegenusDinophysis(Reguera

&Gonza´lez-Gil,2001).Presenceofrecentlydividedcellslacking partoftheleftsulcallistwasalsodetectedinourfieldsamples, whichisasignofrecentcellulardivision.Inourmaterialallthese formswerecommonlyobservedaspartofD.tripospopulations, andtheircharacteristicswereinaccordancewiththosepreviously described from culture and field samples by Rodrı´guez et al.

(2012).However,intermediateandsmallcellsrepresentedonlya smallpercentageoftotalabundance(0.18–2.54)whencompared withothervaluesobservedinDinophysisspp.(10–45%)byReguera

&Gonza´lez-Gil(2001).

5. Conclusion

ThisisthefirstrecordofassociationbetweenDinophysistripos andtoxins inArgentineSea,whereDSPeventsanddetectionof Fig.7.ImageofNoctilucascintillans(length:300mm)(20)containingaD.tripos

partiallyemptythecae.

DTX-1andOAhavebeenpreviouslylinkedtoDinophysisacuminata andDinophysiscaudata(Saretal.,2012).Pectenotoxin-11andPTX- 2saarefoundforthefirsttimeinthisarea.Therecurrentfindingof differentPTXbutlackofOAoritsanalogsagreeswithresultsfrom theBaltic Sea (Seta¨la¨ et al., 2011) and from theChilean coast (Blancoetal.,2007;Krocketal.,2008).

Funding

This workwaspartiallyfinanced bytheHelmholtz-Gemein- schaft Deutscher Forschungszentren through the research pro- grammePACESoftheAlfredWegenerInstitut-HelmholtzZentrum fu¨r Polar- und Meeresforschung and the binational project MINCyT-BMBF (AL/11/03-ARG 11/021), and supported by PIP 00173(CONICET)grantandbytheEuropeanCommissionunder the7thFrameworkProgrammethroughtheAction–IMCONet(FP7 IRSES,ActionNo.319718).

Acknowledgements

TheauthorsthankSebastianGo¨llerforplanktonsamplingand processingduringexpedition2andWolfgangDrebingforsample extraction and toxin measurements. In addition, the friendly receptionandsupportofthe2012crewoftheR/VPuertoDeseado (CONICET-MINDEF, Argentina) and the 2013 crew of the R/V

‘‘Bernardo Houssay’’ (Prefectura Naval Argentina) are acknowl- edged.[SS]

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